Analysis of Amino Acid Changes in Fusion protein of Virulent Newcastle Disease Virus from vaccinated poultry in Nigerian Isolates

doi:10.21203/rs.3.rs-1682034/v1

The roles of Fusion gene in the virulence of Newcastle disease virus has been well established but the extent of its variation among the newly identified XIV, XVII and XVIII genotypes which are predominantly found in Central and West Africa have until recently been understudied and LaSota vaccine strain protection against mortality and morbidity against XIV genotype is the least reported. In this study, F gene of vNDV isolated from samples collected in Nigeria from chicken cadaver from vaccinated chicken flocks showing high mortality, clinical signs and post-mortem lesions attributable to ND during March and April, 2020 were sequenced and analysed for information about genetic changes. Results showed that all isolates from our study have virulent cleavage site sequence 112-RRRKR-116/F117 and clustered within genotype XIVb. Sequence analysis show K78R mutation in the A2 antigenic epitope in all isolates, and more along the F gene which vary in some instances within the isolates. Mutation in this A2 antigenic epitope has been reported to induce escape mutation to monoclonal antibodies generated using NDV LaSota strain. Averages of antigenic variability and percentage homology between the isolates and commonly used vaccines is 0.24; 81.40% and 0.22; 81.90% for LaSota and Komorov vaccines respectively. Nucleotide sequences was assigned accession numbers OK491971-OK491977. Many substitutions were observed in the isolates and some of their functions are yet to be determined.

Virulent NDV

Mutation

Vaccinated poultry

F-gene

Nigerian isolates

amino acid.

Newcastle disease (ND) is a globally reported viral disease affecting over 200 species of birds^[1] primarily controlled by vaccination ^[2]. It is an Organization International des epizootics (OIE) notifiable disease ^[1], however, only a few country report its incidence to OIE, especially in developing countries where the disease is enzootic ^[3] among vaccinated and unvaccinated poultry. ND has received extensive attention because of its ability to spread, high mortality, vaccine failure and other economic losses associated. The aetiology of ND is virulent strain of Avian Paramyxovirus-1 (APMV-1) also known as Newcastle disease virus (NDV) of the genus Orthoavulavirus belonging to the family Paramyxoviridae and order Mononegavirales^[1]. It is a negative sense, non-segmented, single stranded enveloped RNA virus ^[4].

NDV F glycoprotein mediates fusion between the viral and host cellular membranes ^{[5, 6]}. It is synthesized as an unreactive F0 precursor, containing 1662 nucleotides (nt) coding for 553 amino acids (aa) with an approximate 55kDa weight ^{[7, 8]}. F0 is proteolytically cleaved by specific host cellular proteases at the peptide bond between residues 116 and 117 forming two disulphide linked polypeptides; F1 and F2 which are 48–54 kDa and 10-16kDa respectively^{[9, 10]}. There are several domains that have been identified along these polypeptide important for viral fusion activity ^[11].

Although Newcastle disease (ND) is said to be enzootic in Nigeria, little information exists on the molecular epidemiology and the lineage distribution of the Newcastle disease virus (NDV) in the country and there is paucity on reports of virulent Newcastle disease virus (vNDV) strains obtained from sick vaccinated animals in the out-patient veterinary clinic. The importance of detection and pathotyping of NDV in understanding the epizootiology of the virus in any region cannot be overemphasized ^[12] especially with the growing need for evaluation of the efficacy of existing ND vaccines ^[13].

The aim of this study was to isolate and characterize Newcastle Disease Virus (NDV) full Fusion (F) gene obtained from sick vaccinated commercial poultry attending a veterinary clinic in Kano State, Nigeria during March and April, 2020 and analyse amino acid substitution among the isolates.

Animal Ethics Declaration

This study did not include the use of live chickens. International and national guidelines for the care and use of animals were followed by experts at the veterinary clinic during post-mortem examinations and sample collection.

Sample collection

Pooled organ samples (proventriculus, spleen and small intestine) and swabs (Cloacal and tracheal) was collected in viral transport medium (VTM) from one hundred chicken cadaver with history of vaccination against NDV that was reported to a veterinary clinic located in Kano state metropolis during March-April 2020 from flocks presenting with respiratory discomfort, weakness, greenish diarrhoea, anorexia, high mortality and morbidity and drop in egg production in layers characteristic of ND. The samples were labelled and transported immediately on ice and stored at -4⁰C until analysis was conducted.

Post mortem examination was conducted on all specimen, characteristic lesions were noted.

NDV total viral RNA extraction, reverse transcription polymerase chain reaction, F-gene sequencing

All experiments were carried out according to standard protocol. Viral RNA were extracted directly from pooled organ samples and swabs using the Quick-RNA^TM Viral Kit (Zymo Research, USA) as specified in the product manual. Fifty samples were processed successfully.

NDV M-gene was detected using protocols described ^[14]. Two overlapping fragments covering 1662 bp of the full F-gene were amplified using two pairs of primers (Table 1). One-step RT-PCR was performed using One Taq one-step RT-PCR Kit (New England BioLabs ^Inc). cDNA synthesis was achieved at 50^0C for 30 minutes followed by incubation at 94^0C for 15 minutes. RT-PCR was performed with 40 cycles of denaturing at 94^0C for 30 seconds, annealing at 55^0C for 1 minute and extension at 68^0C for 2 minutes with final extension at 68^0C for 10 minutes and PCR products was maintained at 4⁰C. Electrophoresis of PCR products was done on 1% ethidium bromide stained 1.5% agarose gel at 90 volts, 120 Amps for 35 minutes and compared with a 100bp DNA ladder, amplified products were visualized under ultraviolet (UV) illumination using gel documentation system-image capture (Biometra, Germany).

The amplicons was sequenced by Macrogen Ltd (Netherlands), sequences obtained was submitted on the NCBI database with accession numbers OK491971- OK491977.

Phylogenetic tree construction and evolutionary distance analysis

Nucleotide sequence alignment, editing and analysis were done using the Bioedit software (7.2.5). Sixty-two representative NDV F-gene sequences of different genotypes and sub-genotypes majorly those isolated from Nigeria and Africa were downloaded from the Genbank. Nucleotide sequence similarity was calculated using the ClusterW mutilpe alignment algorithm on Bioedit (7.2.5) and MEGA (11.0.). A phylogenetic tree was constructed and evolutionary distance between genotypes and sub-genotypes analysed using MEGA (11.0.) software using the Neighbour joining method with 1000 bootsrap value.

Table 1: Primers used for sequence of Full F-gene of NDV

Primer Name	Direction	Primer	Location	Product Size	References
NDV-F4217F	Forward	5’-TGCGGAGTGTGAAAGTCATCATT-3’	4217-4239	1240bp	JF966385.1
NDV-F5457R	Reverse	5’-TGCTGAGGCAAACCCTTTGT-3’	5438-5457
NDV-F5296F	Forward	5’-ATTGGTAGCGGCTTGATCACTG-3’	5296-5317	999bp
3’NDV-F6295R	Reverse	5’-CGTTCTACCCGTGTACTGCTCTTT-3’	6272-6295

Characteristic pathological lesions on organs seen during post mortem examination

Molecular detection of NDV

The overall M-gene detection rate by RT-PCR was 54% (27/50). A 121-bp fragment was amplified and products of electrophoresis visualized by Ultraviolet (UV) trans-illumination (Fig 1).

F-gene amplification was successfully carried out in seven samples and products of electrophoresis visualized by UV trans-illumination (Fig 2,3). 1662bp nucleotide sequences obtained were deposited in the GenBank and accession numbers assigned (Tab 2).

Tab 2: Sample collected, Sequence ID and corresponding Accession Number

Sample collection ID	Sequence ID	Isolate specimen voucher	Isolated From	Accession Number
PCR/014/200320	Seq 14	F gene KN 14	Broiler	OK491971
PCR/036/250320	Seq 36	F gene KN 36	Broiler	OK491972
PCR/048/300320	Seq 48	F gene KN 48	Broiler	OK491973
PCR/055/310320	Seq 55	F gene KN 55	Broiler	OK491974
PCR/056/310320	Seq 56	F gene KN 56	Broiler	OK491975
PCR/071/020420	Seq 71	F gene KN 71	Layer	OK491976
PCR/075/030420	Seq 75	F gene KN 75	Layer	OK491977

Phylogenetic tree and evolutionary distance analysis

Phylogenetic tree was constructed (Fig 4) using MEGA (11.0). The genetic relatedness of the isolates, a Lasota reference strain KU665482 and other NDV genotypes of known F gene sequences obtained from GenBank database was inferred by phylogenetic analysis. All isolates clustered around the newly classified genotype XIV (sub genotype XIVb) in class II which is widely reported in Nigeria.

Nucleotide blast analysis shows a 99% nucleotide identity to virulent NDV MT543153 isolated in 2019 from backyard poultry in Niger (a country to the North border of Nigeria). KY171993 isolated from Nigeria in 2009 shows a homology 97.65% (as at 16^th February, 2022; data not shown). The nucleotide sequence homologies between these isolates and the common vaccine strains used in the country is shown (Tab 3).

Tab 3: Estimates of evolutionary divergence and percentage homology between study isolates and commonly used vaccines in Nigeria

	KU665482 NDV LaSota strain^a		KT445901 NDV Komorov strain^a
Isolate^a	Genetic distance^b	% Homology^c	Genetic distance^b	% Homology^c
OK491971	0.23	81.44	0.22	81.86
OK491972	0.22	81.56	0.22	81.98
OK491973	0.23	81.50	0.22	82.04
OK491974	0.23	81.44	0.22	81.86
OK491975	0.29	81.44	0.22	81.86
OK491976	0.23	81.26	0.22	81.76
OK491977	0.23	81.20	0.22	81.65

^acalculated from the complete F gene sequences

^bThe number of base substitutions per site from between sequences are shown. Analyses were conducted using the Maximum Composite Likelihood model [17]. This analysis involved 9 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All positions with less than 95% site coverage were eliminated, i.e., fewer than 5% alignment gaps, missing data, and ambiguous bases were allowed at any position (partial deletion option). There were a total of 1662 positions in the final dataset. Evolutionary analyses were conducted in MEGA11 [18]

^cAlign sequence nucleotide blast showing homology of a 99% query cover on NCBI blastn suite

Molecular Characterization and Mutational Analysis of the Functional and Antigenic domains

The complete translated 553 fusion protein amino acid sequences obtained from the study isolates was used to compare their functional and antigenic domains relative to LaSota vaccine strain. Notable substitutions around these regions were observed. Interestingly, among the research isolates, these substitutions were sometimes not observed similarly. Numbering system of aa was used to name the detected aa substitutions with respect to observed genetic variations.

Along the hypervariable region (residue 1-31), 14 substitutions were observed (Fig 5). In comparison to the La Sota reference strain KU665482, all isolates displayed a S31P in the signal peptide region as a result of TCC to CCC substitution at the 91-93. This mutation was seen only in KJ958914 and KJ958913 (obtained from GenBank) among the 69 isolates included during the alignment and phylogenetic study (supplementary document). Isolate OK491976 exhibited unique aa substitutions AGA to GAA, GCA to GAA and CTG to CAG at codons 10-12, 31-33 and 43-45 respectively leading to Arginine to Glutamate at position 4, Alanine to Glutamate at position 11 and Proline to Glutamine at position 15. In contrary, all other study isolates exhibited AGA to AAA and GCA to GTA substitution at codons 10-12 and 31-33 leading to Arginine to Lysine at position 4 and Alanine to Valine substitution at position 11 respectively while the proline at position 15 is conserved. P28 is conserved in OK491972 but not in the other six isolates with CCG to CTG substitution at codons 82-84 leading to Proline to Leucine substitution. A29 was conserved in OK491971 and OK491975 but substituted in the other research isolates due to GCA to ACA substitution at codons 85-87 leading to Alanine to Threonine substitution.

Eight transmembrane domains have been reported ^[19] at residues 14-27, 15-25, 118-131, 120-128, 266-269, 429-432, 499-525 and 501-523. Compared to the LaSota strain, this region is highly non-conserved in all isolates except for 266-269 with no amino acid substitution (Fig 5,6).

All seven isolates share the characteristic virulent motif ¹¹²R-R-R-K-R/F¹¹⁷at the F0 cleavage site indicating that they are velogenic NDV strains (Fig 5). GGC to AGA substitutions at codons 334-336 causing a Glycine to Arginine substitution at position 112; there was AGA to CGA substitution at codon 337-339 in all isolates though Arginine residue is present in both LaSota and research isolates at position 113; CAG to CGG substitution in codon position 340-342 causing a Glutamine to Arginine substitution at position 114; Glutamine to Lysine substitution at position 115 occurred in all isolates, however, isolates OK491973 and OK491976 exhibit GGC to AAA substitution at codons 343-345 while the others show GGC to AAG substitution. At position 116, both LaSota and research isolates exhibit Arginine residue however, aa CGC and CGT is present at codon 346-348 in Lasota and research isolates respectively. CTT to TTT substitution at codon 349-351 occur leading to Leucine to Phenylalanine substitution at position 117 in all the isolates.

Along the fusion peptide region (117-142), five aa substitutions is seen. While L117F and I118V is expected in the virulent furin-like molecule, the I121V, G124S and I135V substitutions are not. This substitutions is due to GGT to AGT nt substitution at codon 361-363; GGT to AGT substitution at codon 370-372 and ATA to GTA substitution at codon 403-405 respectively.

In addition, the F protein has six highly conserved potential N-linked glycosylation sites Ng₁-Ng₆ ^[20] with sequence Asn(Asparagine)-X-Ser(Serine)/Thr(Threonine) (N-X-S/T) where X is any aa except proline and aspartate ^[19,21]. Amino acids at these sites were used and conserved in all NDV isolates of this research at residue 85NRT, 191NNT, 366NTS, 447NIS, 471NNS and 541NNT. Hence, no loss of glycosylation site though there was one substitution compared to the LaSota strain at residue 191NKT. In addition, same sense point mutations occurred at some points as; Asparagine at residue 85 and Arginine at residue 86 show nt mutation but no aa mutation from AAC to AAT (Asparagine) and AGG to AGA (Threonine) in isolates compared to LaSota strain in codon 253-255 and 256-258 respectively. Similarly, at codon 1096-1098, nt substitution from AAT to AAC is observed only in one isolate OK491977 and TCG to TCA substitutions in all isolates at codon 1102-1104, nevertheless, both substitutions did not alter the aa residue Asparagine and Serine at positions 366 and 368 respectively. Asparagine residue at 472 exhibited nt substitution AAC to AAT at codon 1414-1416 while the Serine residue at 473 showed nt substitution only in one isolate OK491977 from TCG to TCA. There was nt substitution from AAT to AAC in the codon 571-573 of the Asparagine residue at position 191. AAA to AAT nt substitution at codon 574-576 cause a Lysine to Asparagine substitution at residue 192. There is nt substitution from ACA to ACG at codon 577-579 of the Threonine residue at 193. All positions 447NIS were all conserved with no nt substitution compared to LaSota. Lastly, the Threonine residue at 543 show nt substitution from ACT to ACC.

Cysteine residues are important in the connection between F1 and F2 subunits. The conserved cysteine in the C-terminal has been reported to maintain the F protein structure. Cysteine residues conserved at positions 25, 27, 76, 199, 338, 347, 362, 370, 394, 399, 401, 424, 514 and 523 of the F protein in most NDV isolates^[21]. Amino acids are used and conserved in all the isolates except for a unique point cysteine (C) to Serine substitution at residue 394 in OK491977. No nt substitution was observed in the Cysteine residues at position 25 (codon 73-75), 76 (codon 226-228), 199 (codon 595-597), 347 (codon 1039-1041), 370 (codon 1108-1110), 399 (codon 1195-1197), 401 (codon 1201-1203), 424 (codon 1270-1272), 514 (codon 1540-1542) and 523 (codon 1567-1569). Nucleotide substitutions was observed at codon 79-81from TCT to TGC of the Cysteine residue at position 27 only in isolate OK491974, nt substitution of cysteine residue 338 from TGT to TGC at codon 1012-1014, TGC to TGT substitution at codon 1084-1086 at Cysteine residue 362, nt substitution at Cysteine residue 394 in OK491977 from TGC to AGT at codon 1180-1182 resulted in a Cysteine to Serine substitution while TGC to TGT (Cysteine) was observed in all the other isolates. Therefore one Cysteine residue site is lost in isolate OK491977.

The major epitopes involved in virus neutralization are conserved in all residues except for one amino acid substitution Lys AAG to Arg AGA (K78R) of the A2 neutralizing epitope identified in all isolates (Fig 5). However, nt substitution occurred even in the conserved epitopes as; Aspartate residue on position 72 show nt substitution from GAT to GAC at codon 214-216 in all isolates; Glutamate residue on position 74 show nt substitution from GAG to GAA at codon 220-222 in all isolates; Alanine residue on position 75 show nt substitution from GCA to GCG at codon 223-225 in all isolates; there was nt substitution from AAA to AGA at codon 232-234 in all isolated that lead Lysine to Arginine amino acid substitution in all isolates and the loss of the A2 neutralizing epitope; at codon 235-237, there is no nt substitution in the Alanine residue. Only 4 nt substitutions is observed along the antigenic epitope 151-171; codon 457-459 nt substitution from CGA to CGG of the 153-Arginine residue; codon 463-465 substitution from AAA to AAG of the 155-Lysine residue; codon 487-489 substitution from GAG to GAA of the 163-Glutamate residue; codon 505-507 is conserved in isolate OK491975 but nt substitution is seen in the others from ACT to ACC of the 169-Threonine residue.

The three Heptad Repeat regions HRa (143-185), HRb (268-299) and HRc (471-500) in the isolates displayed 1, 5 and 6 aa substitutions respectively compared to the LaSota reference strain. K145N at HRa; N272Y, S278P, I285K, T288N, N297K at HRb and N476T, N479D, E482A, R486N, K494R and T498S. Notably, substitutions I285K was seen only in OK491973 and OK491975 only. N297K was seen only in isolate OK491973 only. N476T was observed only in isolate OK491976. Interestingly, T498S was observed in all research isolates but not in any other isolate included in the phylogenetic tree analysis even those isolated previously from Nigeria or Africa (Fig 6, supplementary material).

There is 14 nt substitutions along the HRa region; GCC to GCT at codon 430-432 of the 144-Alanine residue; AAA to AAT (OK491971 only) and AAA to AAC in codon 433-435 lead to Lysine to Asparagine substitution at residue 145; CAA to CAG nt substitution in all isolates at codon 436-438 of 146-Glutamine residue; GCT to GCC substitution at codon 442-444 of 148-Alanine residue; GCA to GCT nt substitution at codon 445-447 of Alanine-149 in isolates OK491945 and OK491947 only; AAC to AAT substitution at codon 448-450 of 150-Asparagine in OK491971, OK491972, OK491974, OK491975 and OK491977 only), CGA to CGG nt substitution is seen in all isolates at codon 457-459 of 153-Arginine; CTT to CTC at codon 460-462 of the 154-Leucine residue; AAA to AAG nt substitution in codon 463-465 of the 155-Lysine residue; GAG to GAA nt substitution in 487-489 of 163-Glutamate residue; ACT to ACC nt substitution of codon 505-507 of 169-Threonine residue; TCG to TCA nt substitution in codon 517-519 of 173-Serine residue in isolates OK491974 and OK491975 only; GCG to GCA in codon 532-534 of 178-Alanine residue and GTT to GTC in codon 535-537 of 179-Valine residue is seen in all isolates.

Eighteen nt substitutions occur along the HRb region; TTA-TTG in codon 802-804 of the 268-Leucine residue; AAC-TAC in codon 814-816 that cause aa substitution Asparagine to Tyrosine at residue 272; CCT-CCC nt substitution in codon 817-819 of the 273-Proline residue; ATT-ATA nt substitution in codon 820-822 of the 274-Isoleucine residue; CTA-CTG nt substitution in codon 823-825 of 275-Leucine residue; TAC to TAT nt substitution in the codon 826-828 in the 276-Tyrosine residue; TCA to CCA nt substitution in codon 832-834 cause aa substitution form Serine to Proline at position 278; CAG to CAA nt substitution in the codon 835-837 of 279-Glutamine is seen only in isolates OK491973 and OK491976; CAA to CAG substitution in codon 841-843 of the 281-Glutamine residue is seen in OK491971, OK491972 and OK491976 only; GGT to GGC nt substitution in codon 850-852 in the 284-Glycine residue; ATA to AAA nt substitution in codon 853-855 is seen in OK491973 and OK491975 only resulting to aa substitution from Isoleucine to Lysine at position 285; CAG to CAA nt substitution in codon 856-858 in the 286-Glutamine residue in all isolates except OK491973; ACT to AAT nt substitution in codon 862-864 cause aa substitution from Threonine to Asparagine at position 288; CTA to TTA nt substitution is seen in codon 865-867 in the 289-Leucine residue; CCT to CCC in the codon 868-870 of the 290-Proline residue; TCA to TCG nt substitution in the codon 871-873 of the 291-Serine residue is seen in all the isolates except OK491974; GTC to GTT in the codon 874-876 of the 292-Valine residue; AAT to AAC nt substitution is seen in the codon 889-891 in 297-Asparagine in OK491974, OK491976 and OK491977 only, whereas, AAT-AAA nt substitution in the same position is seen in OK491973 only causing Asparagine to Lysine aa substitution in position 297.

Along the HRc region, there are 17nt substitution. AAC-AAT nt substitution in the 1414-1416 codon of the 472-Asparagine residue; TGC to TCA nt substitution in the 1417-1419 codon in the 473-Serine residue in isolate OK491977 only; ATC to ATA and AGT to AGC nt substitution occur in the 474-Isoleucine and 475-Serine residues respectively; AAT to ACT nt substitution in the 1426-1428 is seen in isolate OK491976 only causing Asparagine to Threonine aa substitution at position 476; TTG to CTG nt substitution occur in the 1432-1434 478-Leucine residue; AAT to GAC in codon 1435-1437 cause aa Asparagine to Aspartic acid substitution at position 479; TTA to TTG nt substitution in the 1441-1443 in the 481-leucine residue; in codon 1444-1446, GAG to GCA cause Glutamic acid to Alanine aa substitution at position 482; AGC to AGT nt substitution in the 1450-1452 of 483-Glutamic acid residue; AGA to AAC nt substitution in codon 1456-1458 cause aa substitution Arginine to Asparagine in position 486; AAA to AAG nt substitution in the 1459-1461 codon of the 487-Lysine residue; AAA to AGA nt substitution in the 1480-1482 codon cause aa Lysine to Arginine substitution in position 494; CTG to CTA, ACT to AACC, AGC to AGT nt substitutions in the codon 1483-1485, 1486-1488 and 1489-1491 of the 495-Leucine, 496-Threonine and 497-Serine; ACA to TCA substitution in the 1492-1494 codon cause aa Threonine to Serine at position 498.

There are thirteen effective B-cell epitopes for LaSota vaccine as surface-exposed amino acids which were speculated to cause antigenic difference between the vaccine and wild strains ^[19]. Amino acid sequence analysis of the isolates revealed multiple substitutions within these regions (Fig 4).

NDV management in Africa is complicated ^[22]. The economic impact of ND among vaccinated and unvaccinated commercial and backyard poultry in Nigeria is significant. This translates even to point of sale of live birds (the sale of poultry product in Nigeria is unregulated and open) where marketers record death of chickens and other birds due to disease in live bird markets. Research focus has largely been on NDV isolated from wild birds and their role in the epidemiology of the disease and molecular analysis using the full F-gene for pathotyping and lineage distribution studies. However, despite mass administration of vaccines, there is reports of vaccine failure and high mortality among vaccinated flocks ^[23], sub-optimal protection levels among vaccinated flocks ^[24], viral evolution and identification of new genotypes ^{[22, 19]}, significant antigenic distance between circulating and vaccine strains ^[25]. Despite huge patronization of veterinary clinics (for drugs and vaccines) and consultancy by poultry producers, to the best of our knowledge, this is the first report of isolation of vNDV from vaccinated poultry attending a Veterinary clinic in Nigeria.

Seven genotypes (I, II, IV, VI, XIV, XVII and XVIII) have been reported to circulate in Nigeria demonstrating high genetic diversity of NDV for one country ^[26]. A phylogenetic tree was constructed based on the full F gene of the research isolates to determine their genotype. Result suggested that the isolates belong to genotype XIVb in class II similar to earlier reports of newly classified genotypes XIV, XVII and XVIII identified as the circulating strains in Central and West Africa ^{[22, 3, 25]}. The NDV isolates reported here originated from different poultry farms within and outside the metropolis of Kano state. Post mortem lesions include haemorrhagic intestinal ulcers, haemorrhagic caecum tonsils, haemorrhagic and inflamed proventriculus, haemorrhagic trachea among others (Plate i-ix). These lesions agree with the most common overt clinical signs the sick chickens exhibited as difficulty in breathing, greenish diarrhoea, weakness and anorexia with no marked neurological symptoms. Haemorrhage at the tip of the proventriculus is highly suggestive of ND ^{[27, 28]}. Generally distributed lesions suggests the virus is able to infect and replicate in most organs, typical of vNDV as supported by the cleavage site motif of the isolates.

Previous report in a review, phylogenetically, the Nigerian genotype XIVa isolates form a cluster with some strains in Niger Republic while genotype XIVb isolates tend to be more closely related to the 2009 isolates from Benin Republic ^[25]. They further stated that the isolates in genotype XIVa that share the highest nucleotide similarity with those from Niger Republic were all obtained from Sokoto State which shares a direct international border with Niger Republic. In Contrary, the research isolates reported here show 99% homology with MT543153 (obtained on GenBank as at 15th February, 2022) isolated in 2019 from Niger Republic. It has been reported that importation of poultry in Niger Republic is an informal sector with porous borders between Nigeria and Burkina Faso that allow for the entry of live poultry (chicken and guinea fowl) without prior registration and customs clearance. Informal exportation of poultry from Niger is primarily to Nigeria ^[29]. This close phylogenetic relationship can be explained by the cross border movements between the two countries which may facilitate the spread of the virus. Selling of live birds is very common in West Africa, and birds may sometimes be transported hundreds of kilometers between their breeding, rearing, slaughtering, and consumption places. Seasonal high demand and movement also coincide with increased incidence of NDV in Nigeria ^[30] and may be compounded by environmental factors^[31]. Sub-genotype XIVb have been isolated in a commercial farm in Nigeria that have vaccinated with unspecified vaccine strain against NDV ^[22]. With recent isolation of genotype XIVb among flock vaccinated with LaSota and Komorov vaccine strains, the spread of this genotype to other regions of the continent has a potential to spread vaccine resistance as well.

The F protein is capable of provoking host immune response and it is necessary for producing neutralizing antibodies against NDV induced by vaccines ^[19]. Mutations along this gene will impact antibody production which will be heterologous even at the level of F-gene^[32]. Averages of antigenic variability and percentage homology between the isolates and commonly used vaccines is 0.24; 81.40% and 0.22; 81.90% for LaSota and Komorov vaccines respectively indicating considerable diversity ^[33]. This is similar to reports by ^[22], based on the analysis of the complete F coding sequences performed in the study, all the virulent NDV types circulating in Nigeria are shown to be distantly related to currently available vaccine strains in the country of which La Sota vaccine is the most commonly used. Existing antigenic variations among West Africa strains and the LaSota vaccine may affect its protective efficacy to confer protection against all West African strains ^[33], in cases where the LaSota vaccine provided protection against clinical disease, it didn’t prevent infection and viral shedding ^[33]. There is growing need for development of antigenically matched vaccines to circulating strains ^[13]. The evolutionary distance between the vaccination strain and the circulating field strain is an important factor in effective disease control since it explains the continuous occurrence of ND outbreaks despite the extensive poultry vaccination programme in Nigeria ^[25].

The fusion protein is a major target for the immune response, immunity raised against this protein is effective in the neutralization of NDV infectivity ^[34]. Neutralizing epitopes are important in forming antigenic epitopes, aa substitution in this region induce the formation of neutralizing escape variants ^{[35, 36, 37, 21]}. The single point K78E mutation seen in all isolates has been previously reported to induce escape mutation against MAbs generated using LaSota strains ^[6]. The role of this mutation in genotype XIVb requires more attention. Though the scope of this research did not involve the generation of K78E mutants and LaSota efficacy, it is notable from samples and data collected that though the flocks were vaccinated, high level of ND is still reported.

Predicted glycosylation sites on the F gene is well conserved in all isolates, hence no expected impairment is expected in viral structure, during viral replication or virulence mediated by differential glycosylation.

Virulent NDV genotype XIVb was detected among vaccinated poultry in Kano State Nigeria. Although caution is warranted in considering genetic distance between NDV vaccines and the challenge virus as the sole cause of the reported cases of vaccine escape in the field ^[38], the isolation and detection of vNDV genotype XIVb among vaccinated flock requires further research and especially the role of the A2 antigenic epitope in inducing antibody escape mutation among that sub-genotype.

F-S, O.O and E, E.E designed the research. F-S, O.O collected samples. R, L.D and F-S, O.O conducted all molecular testing and result analysis. S,O.O and F-S, O.O designed figures and tables. S, O.O, R, L.D and F-S, O.O wrote the manuscript. S, I designed the F-gene primer and protocol used during research. E, E.E; I, H.I and L, S.A supervised the research. All authors reviewed the manuscript.

Acknowledgments

This work was part of the PhD study of O.O. F-S. The authors acknowledge the support and co-operation of the management and technical staff of PHED Agrovet (‘Gidan Kaji’), Kano State during sample collection. We also acknowledge the technical support of Magaji Amadu Senior Medical Laboratory Scientist-Influenza Research Laboratory, Aminu Kano teaching hospital and Umar Aliyu Ahmad; Medical Laboratory Scientist-TB North-West Nigeria reference laboratory during molecular detection analysis.

We also acknowledge Dr Muhammad Bashir Bello; Department of Veterinary Microbiology, Faculty of Veterinary Medicine, Usmanu Danfodio University, Sokoto Nigeria for his technical advice.

Funding

The authors did not receive any funds, grants or any other support

Competing interest

The authors have no competing interests to declare that are relevant to the content of this article.

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Plate i-x are available in Supplementary Files section.

No competing interests reported.

Plate1.png
Plate i; cross-section of dissection of a Layer, Plate ii-iii; haemorrhagic caeca tonsils (Peyer’s patches)Plate iv; ecchymotic haemorrhage on the proventriculus, Plate v; haemorrhagic enteritis, Plate vi; congested and enlarged liver, Plate vii; haemorrhagic intestinal tract, Plate viii; haemorrhages on the trachea, Plate ix; inflamed and haemorrhagic proventriculus, Plate x; inflamed spleen.

Analysis of Amino Acid Changes in Fusion protein of Virulent Newcastle Disease Virus from vaccinated poultry in Nigerian Isolates

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Abstract

Figures

Introduction

Material And Methods

Results

Discussion

Conclusion

Declarations

References

Supplementary

Additional Declarations

Supplementary Files

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Version 1